Goal 1: Monocytes It has been shown in mouse models of chemically induced cancer that Interferons (IFNs) are important in the immune-surveillance and immune-editing of tumors. While IFNs alpha (IFNa) and gamma (IFNg) have been shown to have potent anti-neoplastic and anti-proliferative properties in vitro, they have shown little efficacy in the clinic. Our observations that IFNs play an important role in killing tumor cells in the presence of monocytes suggests that combination therapy of monocytes and IFNs may have a more potent effect than IFNs alone. We and others have shown that IFNs are more potent anti-cancer agents when used in combination with monocytes isolated from the peripheral blood. Based on these studies we have initiated a Phase 1 clinical trial of immune cell therapy using autologous transfer of ex vivo monocytes stimulated with IFNs into the peritoneal cavity of patients who have resistant disease. We are now in the process of defining the mechanism of IFN-induced monocyte killing of ovarian cancer cells. we found that IFNs induce a unique set of genes regulated in monocytes that occurs only with the combination of IFNa and IFNg, and this signature may be driven by the transcription factor MZF1. In order to better mimic the peritoneal microenvironment of ovarian cancer, we developed a new model of growing complex ovarian cancer neoplasms from cell culture lines and primary samples. We are able to image and quantify complex interactions of IFNs-stimulated monocytes and cancer growths, providing a superior model for studying functional proteomic interactions. In addition, I have the ability to examine tissue and peritoneal fluid samples from women on our phase 1 trial receiving intraperitoneal autologous monocytes stimulated with IFNs, providing another platform for defining dual-IFN induced mechanisms of monocyte activity in the context of the complete immune response. Goal 2: ADCC A monoclonal antibody was developed against a semi-purified human membrane protein preparation derived from cancer tissues. The protein preparation was used in previous clinical trials for use as a cancer vaccine, where it was demonstrated to be safe and efficacious. The antibody, 16C3, was shown to react with the immunizing antigen preparation, as well as several human tumor cell lines and tissues from colorectal, pancreas, lung, and ovarian cancer patients. 16C3 did not cross-react significantly with normal human tissues, thus representing a potential therapeutic product. The target of 16C3 was studied and shown to be related to CEACAM-5/6, a member of the carcinoembryonic antigen family of proteins, which has been shown to be associated with several cancer types. Endometrial, breast and ovarian cancer have specifically been found to have increased expression in human tumor samples. In endometrial cancer, 45/88 (51%) of tissue samples show reactivity through immunohistochemistry, 38/72 (53%) of breast, and although 16/129 (12%) of ovarian cancer specimens stain positive in this series, two subtypes, mucinous 15/22 (68%)and signet cell 2/2 (100%) ovarian cancers, shows significant reactivity (50%) in an IHC of ovarian cancer tissue arrays representing over 600 samples. While animal toxicity studies are ongoing by the company Precision Biologics, we will proceed with in vitro investigation of mechanism, and mouse models of efficacy. Goal 3: T cells Mesothelin (MESO) is a 41-kD cell surface glycoprotein that is highly expressed in many human cancers, including high grade serous adenocarcinoma of the ovary (75%), pancreatic adenocarcinoma (85%), triple negative breast cancer (66%), epitheliod mesothelioma (95%) of patients with MESO-expressing malignancies. While the function of MESO on normal cells is non-essential, the expression of MESO on cancer cells may contribute to the pathology of cancer, with higher expression associated with poorer prognosis, increased metastatic spread, and activation of cell growth pathways. A tremendously innovative immunotherapeutic approach is the use of chimeric antigen receptor-modified T cells (CAR). CAR T-cell therapy relies on re-engineering autologous T cells to express a receptor that allows the T cells to recognize tumor cells. A CAR is a recombinant receptor composed of an extracellular antigen-binding domain and an intracellular T-cell signaling domain. When expressed in T cells, CARs redirect the T cells to target the cancer cells that express the targeted antigen in a human leukocyte antigen (HLA)-independent manner. The most widely used method for T-cell modification is viral transduction, integration and expression of a genetic construct that expresses the chimeric receptor. Another approach to the generation of CAR T-cell therapies that may provide potent anti-tumor activity and improve safety and product preparation involves the use of mRNA to modify T-cells. Using mRNA to re-engineer a patient's T-cells to express a tumor-antigen targeted CAR T-cell can be accomplished in a few hours, allowing on-site preparation and deployment to multiple treatment locations. mRNA CAR T-cells have the safety factor of a limited lifespan, with half-life times similar to antibody therapeutics, and lack of rapid immune activation and proliferation, limiting the risk for severe cytokine release side effects. Meso-targeted CAR T-cells using mRNA have demonstrated significant promise in preclinical studies and clinical studies by intratumoral, intraperitoneal and intravenous of routes of administration. We plan to run a phase 1 clinical trial testing the safety of intraperitoneal administration of the CARMA in women with ovarian cancer and peritoneal carcinomatosis.